Methods for rejuvinating red blood cells

ABSTRACT

Methods and a system for transfusing blood to a mammalian subject. The method includes obtaining a volume of donated red blood cells (RBCs), adding an enhancement composition to the RBCs to form a treated blood composition, incubating the treated blood composition to form an incubated blood composition, and administering the incubated blood composition to a patient. The system includes a functionally-closed, sterile Y-type tube set.

INTRODUCTION

The present disclosure relates to methods for transfusing blood tomammalian subjects, and a system for use with the methods.

Transfusion of blood is an important aspect of treating many disordersand injuries, such as treatment of accident victims and during surgicalprocedures. According to current American Red Cross statistics, about 5million people receive blood transfusions yearly in the United Statesalone. A single accident victim can require as many as 100 pints ofblood. Thus, the collection and distribution of blood and blood productsis a vital part of the health care system. Typically, blood is obtainedfrom a donor and then processed and stored; units of stored blood orblood products are then taken from storage as needed and transfused intoa patient in need. In some cases, the blood may be an autologousdonation, where an individual donates blood in expectation of receivinghis or her own blood by transfusion during a medical procedure.

Donated blood is typically processed into components and then placed instorage until needed. Short term storage can be as long as six weeks,although blood or blood components can be frozen and stored for as longas ten years. Unfortunately, the storage of red blood cells (RBCs) isassociated with “storage lesions,” altering their energy production,oxygen delivery capacity, redox status, and structural/membraneintegrity. For example, the concentration of adenine triphosphate (ATP)in stored RBCs decreases over time. Not only is ATP an energy sourceused by cells to catalyze numerous enzymatic reactions, ATP also signalsendothelial cells to release nitric oxide (NO), which is a potentvasodilator. Additionally, the concentration 2,3-diphosphoglycerate(2,3-DPG) within RBCs is significantly reduced after 14 clays ofstorage, and is often undetectable after 21 days of storage. 2,3-DPGenhances the ability of RBCs to release oxygen by interacting withdeoxygenated hemoglobin, decreasing the hemoglobin's affinity foroxygen, and thereby promoting the release of the remaining oxygen boundto the hemoglobin. Therefore, with diminished levels of ATP and 2,3-DPG,an RBC's ability to oxygenate tissue is severely impaired.

To rejuvenate RBCs before administration into a patient, blood can beincubated with an enhancement composition containing materials thatincrease intracellular concentrations of 2,3-DPG and ATP, improving theability of RBCs to oxygenate tissues. Such enhancement compositionstypically comprise one or more active materials such as inosine,adenine, sodium pyruvate and sodium phosphate (dibasic and monobasic). Auseful enhancement composition is rejuvesol® Red Blood Cell ProcessingSolution (rejuvesol® Solution), which has been marketed by CytosolLaboratories Inc. (now Citra Labs, LLC) since 1991.

While such compositions are effective to improve the metabolic activityof RBCs, there remains a need to develop compositions and methods thatimprove efficacy. Moreover, it has been discovered that suchcompositions may have particular utility during medical procedurestreating certain disorders.

SUMMARY

The present technology provides methods for transfusing blood to amammalian subject, including such methods for the treatment of disordersassociated with characterized by reduced tissue oxygenation. The methodscomprise adding a red blood cell metabolic enhancement composition(herein “enhancement composition”) to a volume of red blood cells,incubating the treated blood composition, and administering theincubated blood composition to the mammalian subject. In someembodiments, the incubated blood composition is washed prior toadministration to the subject.

Aspects of the methods, such as the washing and the steps of adding theenhancement composition to the red blood cells, may be performed at thepoint of care for the mammalian subject. Such processes may becontinuous, using a system proximate to the subject at the point ofcare, in fluid communication

Methods can be performed immediately after blood is collected from adonor and prior to preservation, or the method can be performed afterblood is removed from preservation, immediately before administration toa subject. Before the method is performed, the blood can be processed tobecome red blood cell concentrated (RBCC), or leukocytes can be removedto generate leukoreduced RBCs.

The present technology also provides a method for transfusing RBCs to asubject, at a point of care, comprising obtaining a volume of donatedRBCs, adding an enhancement composition to the volume of donated RBCs toform a treated blood composition, incubating the treated bloodcomposition to form an incubated RBC composition, diluting the incubatedRBC composition with wash solution to form a diluted RBC composition,centrifuging the diluted RBC composition, forming a pellet of RBC and asupernatant, and administering the incubated blood composition to asubject. After washing, the washed RBC composition can be centrifuged toform a pellet of RBCs and a supernatant. The supernatant can be removedand the pellet can be resuspended in an additive solution, to form aresuspended RBC composition.

DRAWINGS

FIG. 1 is a diagrammatic illustration of a representative method fortransfusing blood.

FIG. 2 is a diagrammatic illustration of an apparatus for improving themetabolic function of blood.

It should be noted that the figures set forth herein are intended toexemplify the general characteristics of an apparatus, materials andmethods among those of the present technology, for the purpose of thedescription of certain embodiments. These figures may not preciselyreflect the characteristics of any given embodiment, and are notnecessarily intended to define or limit specific embodiments within thescope of this technology.

DETAILED DESCRIPTION

The following description of technology is merely exemplary in nature ofthe subject matter, manufacture and use of one or more inventions, andis not intended to limit the scope, application, or uses of any specificinvention claimed in this application or in such other applications asmay be filed claiming priority to this application, or patents issuingtherefrom. A non-limiting discussion of terms and phrases intended toaid understanding of the present technology is provided at the end ofthis

The present technology relates to methods of processing whole blood or ablood fraction comprising red blood cells (herein, unless specificallystated otherwise, referred to as “blood”) using a composition (referredto herein as an “enhancement composition”). Such compositions maycomprise a rejuvenating material selected from the group consisting ofinosine, adenine, pyruvate, phosphates, and mixtures thereof.

RBC Metabolic Enhancement Compositions

The present technology provides enhancement compositions which restore,increase or otherwise enhance, directly or indirectly, one or moremetabolic functions of red blood cells. As further discussed below, insome embodiments, enhancement compositions increase the production orconcentration of one or more intracellular constituents associated withthe biochemical or biomechanical function of red blood cells, includingoxygen transport, oxygen release or other metabolic parameters whichaffect the ability of blood to oxygenate tissue. As discussed furtherbelow, in some embodiments enhancement compositions increase theintracellular concentrations of adenine triphosphate (ATP) and2,3-diphosphoglycerate (2,3-DPG).

In some embodiments, enhancement compositions of the present technologycan comprise safe and effective amounts of inosine, pyruvate, adenineand phosphate. A “safe and effective” amount of a rejuvenating materialis an amount that is sufficient to have the desired effect on thebiochemical or biomechanical function of RBCs, without undue adverseside effects on the viability of RBCs or other blood components or asubject to whom the RBCs are administered (such as toxicity, irritation,or allergic response), commensurate with a reasonable benefit/risk ratiowhen used in the manner of this technology. The specific safe andeffective amount of the rejuvenating material will, obviously, vary withsuch factors as the metabolic state of the RBCs, the specificrejuvenating material(s) used, the conditions under which the RBCs areprocessed with the rejuvenating material, and physical condition of thesubject to whom the RBCs are administered.

In various embodiments, the inosine can have a concentration of fromabout 25 g/L to about 30 g/L. Preferably, the concentration of inosinecan be about 25.0 g/L, about 25.5 g/L, about 26.0 g/L, about 26.2 g/L,about 26.4 g/L, about 26.6 g/L, about 26.8 g/L, about 27.0 g/L, about27.5 g/L, about 28.0 g/L, about 28.5 g/L, about 29.0 g/L, about 29.5g/L, or about 30.0 g/L. In various embodiments, the pyruvate can have aconcentration from about 5 g/L to about 15 g/L. Preferably, theconcentration of pyruvate can be about 5 g/L, about 6 g/L, about 7 g/L,about 8 g/L, about 9 g/L, about 10 g/L, about 11 g/L, about 12 g/L,about 13 g/L about 14 g/L, or about 15 g/L. In various embodiments, theadenine can have a concentration of from about 0.2 g/L to about 2 g/L.Preferably, the concentration of adenine can be about 0.2 g/L, about 0.3g/L, about 0.4 g/L, about 0.5 g/L, about 0.6 g/L, about 0.7 g/L, about0.8 g/L, about 0.9 g/L, about 1.0 g/L, about 1.1 g/L, about 1.2 g/L,about 1.3 g/L, about 1.4 g/L, about 1.5 g/L, about 1.6 g/L, about 1.7g/L, about 1.8 g/L, about 1.9 g/L, or about 2.0 g/L. The phosphate canbe a mixture of a monobasic monohydrate salt and a dibasic heptahydratesalt. The phosphate can be a salt of sodium phosphate. The ratio ofmonobasic salt to dibasic salt can be from about 1:2 to about 1:3. Morespecifically, the monobasic salt:dibasic salt ratio can be about 1:20,about 1:2.10, about 1:2.15, about 1:2.20, about 1:2.25, about 1:2.30,about 1:2.35, about 1:2.40, about 1:2.45, about 1:2.50, about 1:2.55,about 1:2.60, about 1:2.65, about 1:2.70, about 1:2.75, about 1:2.80,about 1:2.85, about 1:2.90, about 1.295, or about 1:30. The phosphatemixture can have a final concentration of from about 18 g/L, to about 22g/L. More specifically, the concentration of phosphate mixture can beabout 18 g/L, about 18.5 g/L, about 19 g/L, about 19.5 g/L, about 20g/L, about 20.1 g/L, about 20.2 g/L, about 20.3 g/L, about 20.4 g/L,about 20.5 g/L, about 20.6 g/L, about 20.7 g/L, about 20.8 g/L, about20.9 g/L, about 21 g/L, about 21.5 g/L, or about 22 g/L.

In some embodiments, enhancement compositions comprise:

(a) about 27 (e.g., 26.8) g/L inosine;

(b) about 11 g/L pyruvate (e.g., sodium pyruvate);

(c) about 0.7 (e.g., 0.681) g/L adenine; and

(d) about 21 (e.g., 20.8) 1 g/L, phosphate (e.g., a mixture of about6.21 g/L monobasic, monohydrate; and about 14.6 g/L dibasic,heptahydrate).

Preferably, the composition has a pH of from about 6.5 to about 7.5,more preferably from about 6.6 to about 7.4, more preferably from about6.7 to about 7.1, more preferably from about 6.7 to about 7.0. Anenhancement composition useful in the methods of this technology hasbeen commercialized by Citra Labs, LLC (formerly Cytosol Laboratories),Braintree, Mass., under the mark “rejuvesol®”.

Methods of Processing Blood

The present technology also provides to methods of processing wholeblood or a blood fraction comprising red blood cells (herein, unlessspecifically stated otherwise, referred to as “blood”) using anenhancement composition of this technology, as described above. Suchmethods comprise, for example:

1) mixing blood with an enhancement composition; and

2) incubating the mixture of blood and enhancement composition.

The blood treated in the present methods may be obtained from amammalian subject (a “donor”) using methods among those well known inthe art. In various embodiments the donor is a human subject. The bloodmay be allogeneic (i.e., donated by a subject of the same species) orautologous (obtained by the subject to whom the treated blood is to beadministered, such as may be drawn in advance of a surgical procedure).

Processing of blood using an enhancement composition of the presenttechnology may be performed on blood that has been stored or may beperformed on “fresh” blood at a time proximate to the time it iswithdrawn from the donor. (As used herein, a “proximate” time is anytime 24 hours or less after an initial event (e.g., donation of blood),such as concurrent with the event, or 18 hours, 12 hours, 6 hours, 4hours, 2 hours, 1 hour, 30 minutes, 15 minutes, 10 minutes, 2 minutes, 1minute, or less, after the initial event.) The volume of blood can beconcentrated, by removal of at least a portion of blood plasma, toproduce red blood cell concentrate (RBCC). Storage may be, for example,from 1 to 50 days, or longer, as needed. Storage may be at anytemperature and other conditions to as to maintain viability of the RBCsfor clinically acceptable storage period. For example, storage may be ata temperature of from about 1° C. to about 6° C. In other embodiments,the RBCs may be frozen, at a temperature of about −65° C. or lower, withthe addition of conditioning agents that preserve the viability of theRBCs at such low temperatures. Suitable conditioning agents(cryopreservatives) include glycerol. It is understood that suchconditioning agents are to be removed from the RBCs prior toadministration, such as by methods described below for removal ofcryopreservatives prior to administration.

In some embodiments, the volume of blood is leukoreduced prior to use inthe processes of the present technology. Such leukoreduced bloodcontains fewer leukocytes than whole blood, and may, in someembodiments, be essentially free of leukocytes. Other blood componentmay also be removed from leukoreduced blood, either before, during, orafter removal of the leukocytes.

Incubating the mixture is conducted for a time sufficient to allow RBCsin the blood to assimilate components from the enhancement compositionand achieve a desired effect on a biochemical or biomechanical functionof RBCs. Without limiting the scope of methods, composition and uses ofthe present technology, the incubation may increase the metabolicactivity of RBCs (for example, in the glycolysis, pentose phosphate, andamino acid pathways), restoring a metabolic profile in the RBC close tothose in fresh RBC. This restored profile may result in improved RBCfunction in the transfusion recipient including oxygenation of tissues,nitric oxide (NO) production capacity, and reduced risk of adverseeffect from the transfusion. For example, incubation may be conductedfor a time sufficient to increase the concentration of ATP and 2,3-DPGin RBCs. The time may vary, depending on such factors as the desiredlevel of increase of ATP and 2,3-DPG, the use of mechanical agitation,the amount of enhancement composition used, and temperature of the bloodduring incubation. In some embodiments, about 50 ml of enhancementcomposition is added to RBCs derived from up to about 550 ml of wholeblood. Mixing can be performed by swirling, shaking, rotating, oragitating.

In some embodiments, the blood is tested during incubation to determinewhether one or more desired biochemical or biomechanical attributes ofthe RBCs have been attained. Thus, for example, incubation may beconducted until a desired level of ATP, 2,3-DPG, NO or other biochemicalmarker of RBC function is attained, e.g., essentially equal to levelsfound in fresh blood.

Methods for measuring such markers of RBC function useful herein includemethods among those known in the art. For example, 2,3-DPG can bedetected by assay kits that are commercially available. In one suchassay, 2,3-DPG from blood is split by phosphoglycerate mutase (PGM),when PGM is activated by glycolate-2-phosphate, to producephosphoglycerate (PG) and inorganic phosphate (P_(i)). Both 2-PG and3-PG can be formed, but 2-PG is isomerized by PGM to form 3-PG. In thepresence of ATP, 3-PG is converted to 1,3-DPG by phosphoglycerate kinase(PGK). Next, 1,3-DPG is converted to glyceraldehyde-3-P byglyceraldehyde-3-phosphate dehydrogenase (GAP-DH) and the oxidation ofNADH. Glyceraldehyde-3-P is converted to digydroxyacetone-P bytriosephosphate isomerase (TIM), and dihydroxyacetone-P is converted toglycerol-3-P by glycerol-3-phosphate dehydrogenase (GDH) and theoxidation of NADH. The oxidation of the two NADH molecules can bemonitored spectrophotometrically at a wavelength of 340 nm. Wherenecessary, standard curves can be prepared with standard solutions of2,3-DPG. A kit useful in such methods is commercially available fromRoche Diagnostics Corporation—Roche Applied Science (Indianapolis,Ind.), for the determination of 2,3-DPG in blood in the range of0.02-0.15 μmol. In some embodiments, a sample of blood can be takendirectly from a subject, or from a closed system used in a continuousprocess (as described below), for the 2,3-DPG determination. Thedifference between the 2,3-DPG before and after blood has beenrejuvenated can be determined.

In another method, as an alternative to measuring 2,3-DPG, the partialpressure of O₂ gas required to achieve 50% hemoglobin saturation (P₅₀)is measured. For example, the P₅₀ can be measured at a point of care byuse of a GEM Premier 3000 (Instrumentation Laboratory Company, Bedford,Mass.). In particular, a blood sample is mixed with heparin to preventcoagulation. Then, a drop or two of the blood sample is expelled onto agauze pad. The gauze pad is then positioned at the GEM Premier 3000 forthe P₅₀ analysis. This analysis can be performed before and after theblood has been rejuvenated.

ATP can he detected in blood by a bioluminescence assay. One such assayutilizes luciferase (typically recombinant firefly luciferase), which inthe presence of ATP, converts luciferin to oxyluciferin. Theoxyluciferin is produced in an electronically excited state. Therefore,the oxyluciferin releases a photon of light as it returns to its groundstate. To measure the ATP present in blood, a blood sample can be drawnfrom a patient or from tubing containing treated or untreated blood. Asmall sample of the blood can be mixed with a luciferinduciferasesolution. The solution can be a buffer, such as a Tricine buffer, pH7-8, containing luciferin, luciferase, and Mg²⁺. The ATP in the bloodsample will activate the reaction, which results in luminescence. Theluminescence can be detected in a luminometer. Measurements can be takenbefore and after blood has been rejuvenated. Although the reagents forthis ATP assay are individually available through multiple vendors, somevendors offer kits that comprise all the reagents. One such kit is theATP Determination Kit (A22066) from Molecular Probes, Inc. (Eugene,Oreg.: now Life Technologies, Carlsbad, Calif.).

The blood may be incubated with an enhancement composition for apredetermined time, such as from about 30 seconds to about 24 hours. Ifthe blood is to be stored prior to administration, incubation may occurthroughout the period in which blood is stored, as discussed furtherbelow. For example, the blood can be incubated with an enhancementcomposition for from about 5 minutes to about 90 minutes, or from 15minutes to about 60 minutes, at a temperature of from about 1° C. toabout 45° C., or from about 25° C. to about 40° C., such as at about 37°C. In some embodiments, the blood is incubated for about 30 minutes.

Incubation can be performed using a variety of devices and methods amongthose known in the art. For example, blood can be incubated by immersinga bag containing the mixture of blood and enhancement composition in arecirculating water bath. In another embodiment, incubating can beperformed for about 5 minutes to about 60 minutes (such as for about 30minutes) in a thawing device with a bath temperature set at from about25° C. to about 45° C. Thawing devices include ThermoLine Models: MT202,MT204, or MT210 sold by Helmer Scientific, Noblevilles, Ind., USA,(formrerly ThermoGenesis, Corp, Rancho Cordova, Calif., USA). In anotherexample, incubating can be performed by placing the treated bloodcomposition in an insulated box containing about 6 instant hot gelpacks, wherein the temperature within the insulated box is from about25° C. to about 45° C. A device can be placed within the insulated boxto mix the treated blood composition, or the insulated box can berotated in such a way that the treated blood composition is mixed fromwithin.

The treated fresh blood may be stored after incubation, or may beadministered to a mammalian subject in need of blood. Conditions forstorage include those discussed above. In some embodiments, theadministration is at a time proximate to the time when the blood isobtained and treated using methods of the present technology.

In some embodiments, the processed RBC compositions of the presenttechnology are sterilized. Sterilization can be performed by anypharmaceutically acceptable method, including such methods as are knownto those of ordinary skill in the art. Non-limiting examples ofsterilization include heating, autoclaving, irradiation, and filtering.

Methods may further comprise washing the blood after incubation toremove all, or a portion, of the components of the enhancementcomposition. Washing can be performed using methods among those known inthe art, including such methods as are used for processing frozen bloodto remove glycerol prior to transfusion. For example, washing maycomprise adding a wash solution to a liquid volume of RBCs, centrifugingthe resulting mixture to form a pellet of RBCs, and removing thesupernatant. Wash solutions may include, for example, normal saline.Such washing may be performed with devices known in the art, such as theACP® 215 Automated Cell Processor, sold by Haemonetics Corporation,Braintree, Mass. The washing may be performed at a time proximate toadministration of the blood to a mammalian subject.

In some embodiments, methods comprise removing liquid components of theincubated red blood cell composition prior to administration to amammalian subject. Such methods further comprise, for example,centrifuging the incubated red blood cell composition, forming a pelletof red blood cells, and a supernatant; and isolating the pellet of redblood cells. The pellet of red blood cells may then be suspended in anadditive solution, forming a red blood cell suspension that may beadministered to a mammalian subject. Additive solutions include thoseknown in the art suitable for storage or administration of blood,including normal saline. In some embodiments, such methods furthercomprising adding a wash solution, such as normal saline, to theincubated red blood cell composition prior to the centrifuging. Thus,methods of the present technology include those comprising:

-   -   (a) obtaining a volume of red blood cells from a mammalian        subject;    -   (b) adding an enhancement composition to the volume of red blood        cells, forming a treated blood composition;    -   (c) incubating the treated blood composition, forming an        incubated red blood cell composition;    -   (d) centrifuging the incubated red blood cell composition,        forming a pellet of red blood cells, and a supernatant;    -   (e) isolating the pellet of red blood cells; and    -   (f) suspending the pellet of red blood cells in additive        solution, forming a red blood cell suspension.        Optionally, the process may further comprise diluting the        incubated red blood cell composition with wash solution prior to        the centrifuging step.

Methods of the present technology may be continuous, wherein two or moresequential steps (e.g., adding an enhancement solution and incubating)are performed on a flow of RBCs, substantially without interruptionthrough the process steps. Thus, for example, a liquid volume of RBCsmay flow through an apparatus which adds enhancement solution, subjectsthe mixture of RBCs and enhancement solution to isolation conditions(e.g., heating and mixing). Such a flow may then continue in acontinuous manner to further process steps, including addition of awashing solution, centrifugation, and suspension of RBCs. As discussedbelow, such processes may be performed in an automated closed system influid communication with an intravenous catheter.

Methods of Transfusing and Treatment

The present technology relates to methods for transfusing blood to asubject. Such methods comprise any procedure suitable for administeringto a mammalian subject a liquid volume of blood comprising RBCs thathave been processed according to a method of this technology. As furtherdiscussed below, transfusing may be performed pursuant to any medicallyappropriate procedure, such as for the treatment of diseases ordisorders associated with blood loss or reduced blood function. Specificmethods for administration include those known in the art, such asthrough use of an intravenous catheter.

The present technology provides methods where the processing of blood isperformed at a time proximate to the administration of the processedblood. For example, as defined above, such proximate administration ofblood may be performed 1 hour, 30 minutes, 15 minutes, 10 minutes, 2minutes, 1 minute, or less, after incubation of the red blood cells. Insome processes, the methods are “point of care,” wherein the processesof the present technology are performed at a location proximate, such asin the same room (for example, bed side) or otherwise immediatelyadjacent, to the mammalian subject to be transfused with the RBCs. Asfurther discussed below, such point-of-care processes may be performedusing a system comprising an apparatus adapted to perform two or moresequential steps of a process of the present invention, such as thesteps of obtaining blood, adding an enhancement composition, incubating,washing, and administering. In some embodiments, such a system is influid communication with a device, such as an intravenous catheter, forobtaining blood from a subject or administering blood to a subject. Insome embodiments, the RBCs administered are autologous.

As discussed above, a subject in need of a transfusion can have adisorder characterized by reduced tissue oxygenation. Such disordersinclude those wherein when blood flow is fixed, restricted, reduced, orstopped. Furthermore, blood transfusions can be necessary when blood islost though injury, surgery or disease. Subjects and disorders that maybe treated include; subjects with sepsis or septic shock that are anemicand require a blood transfusion; subjects with Upper GastrointestinalBleeding (“UGIB”) that are anemic and require a blood transfusion;subjects subjected to severe trauma that are anemic and require a bloodtransfusion; subjects that are critically ill (adult and pediatric) inan intensive care unit, who are anemic and require a blood transfusion;subjects that under-go open heart surgery and receive a bloodcardioplegia solution to perfuse the heart during hypothermic, ischemiccross-clamp, thus providing better oxygenation of the myocardium duringopen-heart surgery; subjects suffering a stroke, treating ischemic braintissue following a stroke, thus increasing the oxygen delivery capacityof the systemic circulation via exchange transfusion or by directadministration to the ischemic area via arterial catheter or byretrograde perfusion via the venous circulation; subjects undergoingobstetrical complications, subjects with bleeding ulcers; subjects withhemolytic anemia; and subjects with thrombocytopenia.

Further to the discussion above of methods involving centrifugation ofincubated RBC volume, FIG. 1 shows an exemplary method 10 fortransfusing blood to a mammalian subject. In step 12, a volume of bloodis collected from a donor. The volume of blood can be obtained from thesubject to whom the blood will be transfused, i.e. autologous blood, orthe volume of blood can be allogeneic from a suitable donor. In someembodiments, the volume of blood is stored in various additivesolutions, commonly used in the art, at 1° C. 6° C. In some embodiments,the volume of blood is cryopreserved. The volume of blood can beprocessed by concentrating to produce red blood cell concentrate (RBCC),or by removing leukocytes to produce leukoreduced RBCs (LR-RBCs; whereasnon-leukoreduced blood is NLR-RBC). In yet other embodiments, the volumeof blood is treated with a red blood cell (RBC) enhancement compositionbefore being stored. Alternatively, the method 10 can be performed at atime proximate to when the volume of blood is collected.

In step 14, an enhancement composition is added to the volume of RBCs,forming a treated blood composition. The enhancement composition can betransferred through a functionally-closed sterile fluid pathway, such asdescribed below. Next, in step 16, the treated blood composition isincubated, forming an incubated blood composition.

In optional step 18, the incubated blood composition can be diluted withsaline or other wash solution, to form a diluted blood composition. Theincubated blood composition can he transfused within 24 hours of washingor washing.

In step 20, the incubated blood composition from step 16, or the dilutedblood composition from step 18, is centrifuged. Centrifuging results ina pellet of RBCs and a supernatant. The supernatant can be removed. Inoptional step 22, the pellet of RBCs is suspended in an additivesolution, such as saline.

Finally, in step 23, any of the incubated blood composition from step16, the diluted blood composition form step 18, or the resuspended bloodcomposition from step 22 can be transfused to a subject.

Systems

As discussed above, processes of the present technology may be performedin a system apparatus adapted to perform two or more sequential steps ofa process of the present invention in a continuous process. For example,one such system comprises:

-   -   (a) an incubation system, for mixing an enhancement composition        with a volume of RBCs, which may be in fluid communication with        a supply volume of the enhancement solution, and which may        comprise a temperature control device for maintaining the        temperature of the volume of RBCs during incubation;    -   (b) a washing system, for adding a washing solution to incubated        RBCs;    -   (c) a centrifugation system, for isolating RBCs from the liquid        volume of incubated RBCs; and    -   (d) a suspension system for adding a volume of additive solution        to the isolated RBCs, to form an RBC suspension.

In some point of care embodiments, the system is in fluid communicationwith an intravenous catheter or other device by which the RBC suspensionis directly administered to a subject. In alternative, or in addition,the system may be in communication with a second intravenous catheterfor obtaining a volume of whole blood directly from a subject, which maybe the same subject to which the RBC suspension is administered.

In some embodiments, the system is automated, such that one or moresteps of a process of this technology are performed without substantialintervention by a clinical operator during the process steps.Preferably, such systems are “closed,” to minimize or eliminate exposureof the RBCs, the enhancement composition and other compositions used inthe process to microbial contamination.

As discussed above, incubation of the blood may be conducted until adesired level of a biochemical marker of RBC function is attained. Thus,the incubation system may comprise a measurement device by which analiquot of blood may be obtained and tested for the concentration of themarker, and the duration of incubation adjusted accordingly. Forexample, 2,3-DPG can be detected online by injecting reagents for a2,3-DPG assay, such as is available from Roche DiagnosticsCorporation—Roche Applied Science (Indianapolis, Ind.), as describedabove, into a tube of the closed system. The tube can comprise atransparent section, such as made from quartz, which can be positionedin a spectrophotometer. The difference between the 2,3-DPG before andafter blood has been rejuvenated can be determined. In anotherembodiment, ATP can be detected online in a closed system, by injectinga luciferin/luciferase solution (as described above) into a transparentsection of tubing in the system that contains blood. This transparentsection of tubing can be enclosed in a dark box wherein no externallight can enter. A photomultiplier tube (PMT) connected to an externalcomputer is positioned immediately below the transparent tubing withinthe dark box. The PMT can detect light produced by the assay, and acomputer can display the results. Measurements can be taken before andafter blood has been rejuvenated.

FIG. 2 depicts a closed, Y-type tube set 30 system useful in methods ofthe present technology. The tube set 30 includes a vented spike 32 withdrip chamber 34 coupled to the distal end of a first tube 36. Spike 32can be inserted into a blood treatment vial 38 or bottle to complete afluid communication channel between the interior of blood treatment vial38 and first tube 36. The blood treatment vial 38 can include anenhancement composition. Also, a first clip 40 can be coupled to thefirst tube 36 to regulate flow through first tube 36.

First tube 36 is coupled at its second end to an input of a Y-shapedconnector 42. An in-line micro-biotic barrier filter 44 is positioned inthe first tube 36 flow path to filter the material flowing from thevented spike 32 through the first tube 36 to the input of Y-shapedconnector 42. One exemplary in-line micro-biotic barrier filter 44 is aflat 0.2 micron filter.

A transfer bag 46 can be coupled to a first end of a second tube 48. Thesecond end of second tube 48 is coupled to the other input of Y-shapedconnector 42. Transfer bag 46 can be initially empty and used tocollect, for example, supernatant waste material. Additionally oralternatively, transfer bag 46 may initially include a processing agent,such as a wash solution. A second clip 50 can be coupled to the secondtube 48 to regulate flow through second tube 48. Further, a secondtransfer bag 52 can be coupled to the second tube 48 by a third tube 54.The second transfer bag 52 can be initially empty and used to collect,for example, supernatant waste material. Additionally, or alternatively,transfer bag 52 may initially include a processing agent, such as anadditive solution. A third clip 56 can be coupled to the second tube 48to regulate flow through second tube 48.

Fourth tube 58 can be coupled to the output of the Y-shaped connector 42at its first end. A second end of fourth tube 58 is defined by a seal 60sealing the fluid channel of fourth tube 58. For example, seal 60 can bewelded closed or to a fifth tube 62 coupled to a blood bag 64 using aradio frequency (RF) tube sealer (not shown). Additionally, the bloodbag 64 can comprise a sixth tube 66 that can transfer treated blood to asubject. Given that no potential entry point of the Y-type tube set 30is initially unsealed or unprotected by a micro-biotic barrier filter,this Y-type tube set 30 is functionally-closed. In other words, theY-type tube set 30 provides a functionally-closed, sterile fluid pathway(via fourth tube 58) for blood enhancement composition from bottle 38 tobe delivered into the blood bag 64.

Because the blood enhancement composition can be transferred to thevolume of donated blood by a sterile, functionally-closed fluid pathway,the blood can be stored for more than 24 hours after such delivery.Additional post-processing devices can be used to further process theblood while maintaining the blood in a functionally-closed, sterileenvironment. For example, the volume of donated blood can be processedusing a functionally-closed, sterile centrifuge. This can be done, forexample, prior to delivering the enhancement composition to the volumeof donated blood, contained in a blood bag, to remove components ofwhole blood and leave RBCs in the blood bag. One exemplaryfunctionally-closed, sterile centrifuge is the COBE Spectra ApheresisSystem. Post-wash storage beyond 24 hours can be achieved using“functionally closed” blood cell processing systems such as theHaemonetics Blood Cell Processor model ACP 215.

Methods and systems among those of the present technology areillustrated in the following non-limiting examples.

EXAMPLE 1

A Y-type tube set is assembled is assembled as shown in FIG. 2 andconnected to a unit of RBCs. The tube set includes a 0.2 μm filter. RBCenhancement composition (rejuvesol® Solution) is transferred to the RBCsvia a vented spike to form a treated blood composition. The treatedblood composition is mixed and incubated, forming an incubated bloodcomposition. The treated blood composition is centrifuged to form apellet of RBC and a supernatant. The supernatant is transferred to anempty waste bag, and the pellet of RBCs was resuspended in 100 mLadditive solution that was transferred from a second transfer bag.

EXAMPLE 2

A Y-type tube set is assembled as shown in FIG. 2 and connected to aunit of 250 mL RBCs. 100 mL additive solution, comprising saline, wastransferred to the RBCs. The tube set includes a 0.2 μm filter. 50 mLRBC enhancement composition (rejuvesol® Solution) was transferred to theRBCs via a vented spike to form a treated blood composition. The treatedblood composition was mixed and incubated, forming an incubated bloodcomposition. The incubated blood composition was then diluted with 200mL wash solution from a first transfer bag containing 200 mL washsolution, forming a diluted blood composition. The diluted bloodcomposition was centrifuged to form a pellet of RBCs and a supernatant.The supernatant was transferred to the now empty first transfer bag, andthe pellet of RBCs was resuspended in 100 mL additive solution, whichwas transferred from a second transfer bag.

Blood processed according to Examples 1 and 2 are analyzed forconcentrations of rejuvenation materials in the final processed RBCsuspensions, with the following results.

The treatment of stored LR-RBCs with rejuvesol®, either concurrently orpost storage prior to transfusion resulting in greater restoration of2,3-DPG. Even though the pre-treatment packed cell weight of the LR-RBCunits were greater than the NLR-RBC units (Table 1), recent studiesdemonstrate that the invention resulted in significantly higherconcentration of 2,3-DPG to levels in LR-RBC derived from 500 mL ofwhole blood (WB) that were treated rejuvesol® solution when compared toNLR-RBC that were derived from 450 mL of WB (Table 2).

TABLE 2 Pre-rejuvenation Net RBC Weight (grams) LR-RBC Derived NLR-RBCDerived from from 450 mL 350.1 338.1 Mean (n = 14}  18.3  19.5 SD318.9-379.0 305.0-366.5 Range

TABLE 3 Summary; ATP and 2,3-DPG values after rejuvenation,deglycerolization and 24-post deglycerolization LR-RBC NLR-RBC Derivedfrom 500 mL of WB Derived from 450 mL of WB ATP 2,3-DPG ATP 2,3-DPG(μmol/gHbg) (μmol/gHbg) (μmol/gHbg) (μmol/gHbg) 7.94 11.04 7.55 8.13Mean Post-Rej (n = 14} 1.09  1.54 0.73 1.60 SD 5.89-9.60 9.05-13.686.27-8.75 5.75-11.01 Range NS † NS † †p < 0.0001 NS p < 0.27 7.50 16.727.82 13.91 Mean Post Delyc (n = 13} 0.48  1.64 0.68  1.89 SD 6.62-8.9413.67-19.58 6.80-9.20 11.17-17.29 Range NS * NS * *P < 0.0005 (n = 13}NS p < 0.77 7.59 17.73 8.39 15.64 Mean 24-hr Post Deglyc (n = 14} 0.85 2.08 0.91  1.48 SD 6.62-9.05 13.42-20.87 6.76-9.53 12.73-18.59 RangeNS + NS + +p < 0.0058 NS p < 0.77

The embodiments and the examples described herein are exemplary and notintended to be limiting in describing the full scope of compositions andmethods of the present technology. Equivalent changes, modifications andvariations of embodiments, materials, compositions and methods can bemade within the scope of the present technology, with substantiallysimilar results.

Non-Limiting Discussion of Terminology

The headings (such as “Introduction” and “Summary”) and sub-headingsused herein are intended only for general organization of topics withinthe present disclosure, and are not intended to limit the disclosure ofthe technology or any aspect thereof. In particular, subject matterdisclosed in the “Introduction” may include novel technology and may notconstitute a recitation of prior art. Subject matter disclosed in the“Summary” is not an exhaustive or complete disclosure of the entirescope of the technology or any embodiments thereof. Classification ordiscussion of a material within a section of this specification ashaving a particular utility is made for convenience, and no inferenceshould be drawn that the material must necessarily or solely function inaccordance with its classification herein when it is used in any givencomposition or method.

The description and specific examples, while indicating embodiments ofthe technology, are intended for purposes of illustration only and arenot intended to limit the scope of the technology. Moreover, recitationof multiple embodiments having stated features is not intended toexclude other embodiments having additional features, or otherembodiments incorporating different combinations of the stated features.Specific examples are provided for illustrative purposes of how to makeand use the compositions and methods of this technology and, unlessexplicitly stated otherwise, are not intended to be a representationthat given embodiments of this technology have, or have not, been madeor tested. Equivalent changes, modifications and variations of someembodiments, materials, compositions and methods can be made within thescope of the present technology, with substantially similar results.

As used herein, the words “preferred” or “preferable” refer toembodiments of the technology that afford certain benefits, undercertain circumstances. However, other embodiments may also be desirable,under the same or other circumstances. Furthermore, the recitation ofone or more desired embodiments does not imply that other embodimentsare not useful, and is not intended to exclude other embodiments fromthe scope of the technology.

Although the open-ended term “comprising,” as a synonym ofnon-restrictive terms such as including, containing, or having, is usedherein to describe and claim embodiments of the present technology,embodiments may alternatively be described using more limiting termssuch as “consisting of” or “consisting essentially of.” Thus, for anygiven embodiment reciting materials, components or process steps, thepresent technology also specifically includes embodiments consisting of,or consisting essentially of, such materials, components or processesexcluding additional materials, components or processes (for consistingof) and excluding additional materials, components or processesaffecting the significant properties of the embodiment (for consistingessentially of), even though such additional materials, components orprocesses are not explicitly recited in this application. For example,recitation of a composition or process reciting elements A, B and Cspecifically envisions embodiments consisting of, and consistingessentially of, A, B and C, excluding an element D that may be recitedin the art, even though element D is not explicitly described as beingexcluded herein.

As used herein, the word “include,” and its variants, is intended to benon-limiting, such that recitation of items in a list is not to theexclusion of other like items that may also be useful in the materials,compositions, devices, and methods of this technology. Similarly, theterms “can” and “may” and their variants are intended to benon-limiting, such that recitation that an embodiment can or maycomprise certain elements or features does not exclude other embodimentsof the present technology that do not contain those elements orfeatures.

Disclosure of values and ranges of values for specific parameters (suchas temperatures, molecular weights, weight percentages, etc.) are notexclusive of other values and ranges of values useful herein. It isenvisioned that two or more specific exemplified values for a givenparameter may define endpoints for a range of values that may be claimedfor the parameter. For example, if Parameter X is exemplified herein tohave value A and also exemplified to have value Z, it is envisioned thatparameter X may have a range of values from about A to about Z.Similarly, it is envisioned that disclosure of two or more ranges ofvalues for a parameter (whether such ranges are nested, overlapping ordistinct) subsume all possible combination of ranges for the value thatmight be claimed using endpoints of the disclosed ranges. For example,if parameter X is exemplified herein to have values in the range of1-10, or 2-9, or 3-8, it is also envisioned that Parameter X may haveother ranges of values including 1-9, 1-8, 1-3, 1-2, 2-10, 2-8, 2-3,3-10, and 3-9.

When an element or layer is referred to as being “on,” “engaged to,”“connected to,” or “coupled to” another element or layer, it may bedirectly on, engaged, connected or coupled to the other element orlayer, or intervening elements or layers may be present. In contrast,when an element is referred to as being “directly on,” “directly engagedto,” “directly connected to,” or “directly coupled to” another elementor layer, there may be no intervening elements or layers present. Otherwords used to describe the relationship between elements should beinterpreted in a like fashion (e.g., “between” versus “directlybetween,” “adjacent” versus “directly adjacent,” etc.). As used herein,the term “and/or” includes any and all combinations of one or more ofthe associated listed items.

Although the terms first, second, third, etc. may be used herein todescribe various elements, components, regions, layers and/or sections,these elements, components, regions, layers and/or sections should riotbe limited by these terms. These terms may be only used to distinguishone element, component, region, layer or section from another region,layer or section. Terms such as “first,” “second,” and other numericalterms when used herein do not imply a sequence or order unless clearlyindicated by the context. Thus, a first element, component, region,layer or section discussed below could be termed a second element,component, region, layer or section without departing from the teachingsof the example embodiments.

1-25. (canceled)
 26. A method for processing a blood componentcomprising the steps of: adding an enhancement composition to a volumecomprising red blood cells (RBCs) to form an RBC composition, whereinthe enhancement composition comprises inosine, adenine, pyruvate, sodiumphosphate, or a mixture thereof. incubating the RBC composition for aperiod to form a treated RBC composition, including mixing the RBCcomposition during the incubating.
 27. The method of claim 26, whereinthe RBC enhancement composition comprises about 25 g/L to about 30 g/Linosine, about 0.2 g/L to about 2 g/L adenine, about 5 g/L to about 15g/L pyruvate, and about 17 g/L to about 23 g/L sodium phosphate, or acombination thereof.
 28. The method of claim 26, wherein the RBCenhancement composition comprises about 27 g/L inosine, about 11 g/Lpyruvate, about 0.7 g/L adenine, and about 21 g/L sodium phosphate. 29.The method of claim 28, wherein the sodium phosphate comprises about 6.2g/L sodium phosphate, monobasic, monohydrate; and about 14.6 g/L sodiumphosphate, dibasic, heptahydrate.
 30. The method of claim 26, whereinthe incubating is at a temperature between 1° C. and about 45° C. 31.The method of claim 28, wherein the incubating is not in a water bath.32. The method of claim 28, wherein the mixing includes swirling,shaking, rotating, or agitating the RBC composition.
 33. The method ofclaim 28, wherein the period is from about 30 minutes to about 90minutes.
 34. The method of claim 28, wherein the volume comprising redblood cells as stored at a preservation temperature prior to the addingstep.
 35. The method of claim 28, wherein the volume comprising redblood cells has leukocyte reduced.
 36. The method according to claim 28,wherein the treated RBC composition is for autologous transfusion. 37.The method of claim 28, wherein the incubating is for about 60 minutesat a temperature of about 37° C. and is not in a water bath.
 38. Amethod for treating a subject experiencing sepsis, septic shock, UpperGastrointestinal Bleeding (UGIB), anemia, trauma, heart attack orstroke, the method comprising. obtaining a volume of red blood cells(RBCs); adding a solution comprising inosine, adenine, pyruvate, sodiumphosphate, or a combination thereof to the volume of RBCs; incubatingthe RBCs and the solution for a period to form treated RBCs, includingmixing the RBCs and the solution during the period; and administeringthe treated RBCs to the subject.
 39. The method of claim 38, wherein thesolution comprises about 25 g/L to about 30 g/L inosine, about 0.2 g/Lto about 2 g/L adenine, about 5 g/L to about 15 g/L pyruvate, and about17 g/L to about 23 g/L sodium phosphate, or a combination thereof. 40.The method of claim 38, wherein the solution comprises about 27 g/Linosine, about 11 g/L pyruvate, about 0.7 g/L adenine, and about 21 g/Lsodium phosphate.
 41. The method of claim 38, further comprising washingthe treated RBCs prior to the administering.
 42. The method of claim 41,wherein the adding, incubating and washing steps are performed in acontinuous process in a closed system at a point of care.
 43. The methodof claim 38, further comprising measuring the partial pressure of oxygenrequired to obtain 50% hemoglobin saturation (p50) of the treated RBCsbefore the administering.
 44. The method of claim 38, wherein the addingincludes passing the composition through a filter.
 45. The method ofclaim 38, wherein the incubating is for a period of about 30 minutes toabout 90 minutes and at a temperature between about 35° C. and about 45°C.